Bridging racemic lactate esters with stereoselective polylactic acid using commercial lipase catalysis

Article abstract of DOI:10.1039/c3gc41457d

A productive and enantioselective hydrolysis of racemic mixtures of lactate esters with commercial Candida rugosa lipase was performed. This step contributes to a novel envisioned route for stereoselective PLA production by combining recent chemocatalytic developments with this biocatalytic contribution, foreseeing two separate l- and d-lactate enantiomer streams. A study of the hydrolysis kinetics identified an unexpected rate determining step at the origin of an unprecedented ester reactivity order.

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Bridging racemic lactate esters with stereoselective polylactic acid using
commercial lipase catalysis
Pieter Van Wouwe, a Michiel Dusselier, a Aurelie Basiç a and Bert F. Sels*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
A productive and enantioselective hydrolysis of racemic mixtures of lactate esters with commercial
Candida rugosa lipase was performed. This step contributes to a novel envisioned route for
stereoselective PLA production by combining recent chemocatalytic developments with this biocatalytic
contribution, foreseeing two separate Lꢀ and Dꢀlactate enantiomer streams. A study of the hydrolysis
10 kinetics identified an unexpected rate determining step at the origin of an unprecedented ester reactivity
order.
45 lactide. While LꢀLA is the natural isomer in the commercial
fermentation process, DꢀLA is not abundantly available. It comes
at astronomic prices today despite recent efforts and
Introduction
Lactic acid (LA) is a common renewable chemical with multiple
applications in the food, cosmetic and pharmaceutical industry.1 2
15 A particularly fascinating and emerging field is its use as solvent
and building block to prepare chemicals and polymers of
commercial value.3ꢀ7 Polylactic acid (PLA), a polyester which
fulfills the definitions of both “renewable” and “biodegradable”,
has been recognized as one of the most promising bioꢀplastics8ꢀ10
20 with an outspoken potential as sustainable alternative for certain
petroleumꢀbased plastics.4 Next to the commodity polymer
market, PLA is useful in various medical applications such as
drug coatings, sutures and prostheses due to its biocompatibility
and controllable degradability.11 The global PLA production
25 increases annually and is projected to amount up to 800,000 ton
in 2020.12
24
developments.23, The current production cost of LꢀLA is too
high to foresee a global PLLA breakthrough mainly because of (i)
50 the low volume productivity, viz. 0.3 to 5 g/L.h,25ꢀ28 despite its
high yield starting from glucose (around 90%)1, 29ꢀ31(ii) the costly
multiple purification and separation steps of LꢀLA from the
fermentation broth, and (iii) the formation of one ton of gypsum
waste per ton LꢀLA, resulting from acid neutralisation.2, 31 As this
55 salt waste is probably the main bottleneck, recent research was
focused in the development of acid tolerant microorganisms, but
lactic acid yields and productivities are low.31ꢀ33 Another
advancement was the development of electrodialysis membranes,
as a useful tool to eliminate the salt waste, but resulting in an
60 overall more complex process system. The above mentioned
issues might compromise the genuine greenness of LꢀLA
synthesis, especially with the large production scale outlook
ahead. We therefore envision an alternative synthesis route
leading to separate Lꢀ and DꢀLA streams from common sugars by
65 combining the best of chemoꢀ and biocatalysis (Scheme 1).
Commercial PLA production involves ringꢀopening
polymerization of lactide, the cyclic dimeric form of lactic
acid.13, 14 Stereoisomerism, as described first by van ‘t Hoff,15 is
30 paramount to the PLA quality. LA, obtained via bacterial
fermentation of sugar, has the Lꢀconfiguration and as
a
consequence, Lꢀlactide is the building block of commercial polyꢀ
Lꢀlactic acid (PLLA).16, 17 Mesoꢀlactide, having one Lꢀ and one
Dꢀconfiguration, is undesired because its incorporation in stereoꢀ
35 pure PLLA leads to inferior thermal and mechanical properties.18,
19
PolyꢀDꢀlactic acid (PDLA), derived from Dꢀlactide, behaves
identically to PLLA. More importantly, blends of PLLA and
PDLA contain homoꢀstereocomplex forms with a peculiar crystal
structure providing
a
superior thermal and mechanical
40 performance.20ꢀ22 The availability of separate Lꢀ and Dꢀlactide
feed streams is thus of utmost importance, not only for large scale
stereocomplex PLA production, but also to create medical grade
PLA (e.g. for drug delivery) with a controllable hydrolytic
biodegradation rate by applying a controlled ratio of Lꢀ to Dꢀ
Scheme 1 Tentative route for stereoselective PLA by combining a
chemocatalytic production of racemic lactates from sugars with an
enzymatic enantioselective hydrolysis
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The newly proposed strategy involves a sequence of a
chemocatalytic conversion of the sugars to racemic alkyl lactates,
currently under intense investigation,1 followed by an enzymatic
enantioselective hydrolysis. The chemocatalytic route relies on a
resolutions of alcohols, carboxylic acids, esters, and amines56, 57,
73ꢀ77
60
but examples on resolutions of αꢀhydroxy esters are very
rare.78, 79
5
recent breakthrough yielding LA (in water) and its esters (in
Experimental
34ꢀ42
alcohol) from sugars
containing catalysts
in presence of robust, solid Al or Sn
Reactions were carried out in 10 mL thick glass walled crimp cap
vials. In a typical experiment a known amount of ester (Sigmaꢀ
43ꢀ47
36, 39, 48
such as Snβ zeolite
and Sn
49
modified silica/carbon hybrids.46,
This alternative route with
65 Aldrich or derived from lactide) (e.g. 0.4 g in the case of MLA)
and e.g. 0.08 g enzyme preparation (SigmaꢀAldrich) was added
together with 7 mL phosphate buffer (pH = 7.2). A nonꢀ
interfering internal standard (1,4ꢀdioxane (Acros)) was used. A
magnetic stirring bar was added and the vial was closed and
70 placed in a copper heating block at a fixed temperature (e.g. 45
°C). Samples were taken at certain moments in time. Enantiomers
of MLA, ETLA, nPrLA, MHBA, MGA, EGA and nPrGA were
analyzed by chiral gas chromatography (GC) on a 25 m WCOT
fused silica CPꢀChirasilꢀDEX CB (Agilent) capillary column. The
75 Eꢀvalue (enantiomeric ratio) was determined using following
formula: E = ln[1ꢀc(1+eep)]/ln[1ꢀc(1ꢀeep)].80 The synthesis of
lactate esters was performed via alcoholysis of lactides
(generously provided by Purac) as described in the supporting
information.
less workꢀup could be a future commercial route to lactic acid
10 and its esters, provided that one could deal with the racemic LA
mixture. Being racemic, the chemically derived lactate ester feed
indeed has the desired composition to create both Lꢀ and Dꢀ
lactide at comparable prices. However, direct processing of the
racemic mixture with stateꢀofꢀtheꢀart technology would lead
15 mostly to unwanted mesoꢀlactide, apart from equimolar amounts
of Lꢀlactide and Dꢀlactide. Considering the need for pure Lꢀ and
Dꢀlactide streams, a separation of the lactate racemate into pure
enantiomers is compulsory. Chromatographic methods are timeꢀ
consuming and expensive50ꢀ52 and although chemical resolution
20 by reacting lactic acid racemates with Lꢀbrucine is a wellꢀknown
separation technique, it is generally characterized by a low
efficiency.53 More efficient and cheap separation methods should
be applied to utilize the racemic feed as PLA polymer precursor.
Enzymatic kinetic resolution has proven to be successful in
25 obtaining enantiopure compounds.54ꢀ63
80 Results and discussion
Herein we report the enantioselective hydrolysis of racemic
αꢀhydroxy compounds like the aforementioned alkyl lactates with
Candida rugosa lipase (CRL). The racemic ester substrates are
available in high yields via heterogeneous catalysis in alcoholic
30 media (scheme 1), while no catalyst deactivation was observed in
contrast to the chemocatalytic synthesis of LA in water.36, 40, 46
Moreover, the esters are easier to handle in purification
procedures, and in the context of biocatalysis, they offer the
advantage over the free acid that significantly less buffer is
35 needed. These arguments motivate the enzymatic resolution
method by means of ester hydrolysis. Besides, while multiple
reports of kinetic resolutions are focused on the synthesis of one
key enantiomer, the goal here is to directly provide the two
processable streams of opposite enantiomers, only requiring a
40 common ester/acid separation. Ultimately, LꢀLA is further
converted directly to Lꢀlactide with the current process
technology,13 while Dꢀlactate ester is readily hydrolyzed and
further processed similarly or even processed directly to Dꢀ
lactide.64
Screening
In an initial phase, commercial lipase preparations were screened
for their activity and enantioselectivity towards the hydrolysis of
racemic methyl lactate (MLA). The screening was carried out in
85 dilute conditions in analogy with reported procedures.55, 78, 81ꢀ83 In
the mild aqueous conditions, only LA and methanol were
analyzed as product. Reactions were buffered and performed at
room temperature to avoid nonꢀstereoselective background
hydrolysis. Table 1 summarizes the catalytic data of different
90 lipases at nearly the same degree of total ester conversion.
Table 1 Screening of different commercial lipase preparations in the
enantioselective hydrolysis of MLA in water.a
Entry Enzyme Time
[h]
Conv.(L) Conv.(D)
eep
Pur.
[%]d
96.4
[%]b
73.8
[%]b
2.8
[%]c
92.8
1
2
3
4
5
CRL
CALB
PPL
46
2
36.3
60.0
46.0
61.6
42.4
22.0
31.4
19.8
ꢀ7.9
39.7
17.8
45.4
46.1
69.9
58.9
72.7
191
561
224
BCL
LM
45
Lipases are the biocatalyst of choice to assist the
enantioselective hydrolysis step. They have verified timeꢀonꢀ
stream stability, substrate specificity, and high enantioselectivity
in mild reaction conditions with multiple applications in the food,
pharmaceutical and agrochemical industries.58, 62, 65ꢀ67 Moreover,
a
Hydrolysis at 25 °C with enzyme preparation (abbreviations in full in
text) (0.02 g in the case of CRL and CALB; 0.15 g in the case of PPL,
95 BCL and LM), 0.135 M MLA, 7 mL 0.014 M phosphate buffer (pH =
b
7.2). Determined by chiral GC (see experimental and supporting
50 lipases work without cofactors and they are produced
extracellularly. This results in current industrial sales prices
around €500/kg enzym for commercial lipase formulations of
CRL, their production cost typically being a factor two to three
lower.68 With the novel fermentation techniques ahead for lipase
55 production, the enzyme price should not be a show stopper for its
use in stereoselective PLA synthesis (see supporting
information).69ꢀ72 Frequently applied in organic synthesis, lipases
have been demonstrated to be particularly valuable in kinetic
c
d
information). enantiomer excess of LꢀLA on product side. Molar
enantiomeric purity on product side (see supporting information).
100
Candida rugosa lipase (CRL, Entry 1) outperforms all
other enzymes with respect to enantioselectivity. CRL preferably
hydrolyzes the LꢀMLA isomer with a product enantiomeric
excess eep of 93% at very high conversion of the Lꢀisomer of
MLA. This corresponds with a molar enantiomeric purity on the
2
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product side of 96.4%, which is a measure for the ultimate purity
of the corresponding lactides and PLA polymers. Candida
enantioselectivity at 45°C and MLA concentration of 0.55 M or
higher. Surprisingly, no significant reduction in enantioselectivity
antarctica lipase B (CALB, Entry 2), which was used in the 50 was observed for the concentrated conditions, viz. 0.55 M and
immobilized form, has by far the highest activity, but its
enantioselectivity is very poor and slightly in favor of DꢀLA
formation. The three other commercial lipases Porcine pancreas
lipase (PPL, Entry 3), Burkholderia Cepacia lipase (BCL, Entry
1.10 M MLA. These concentrations may be considered very high
and industrially practical,84 when compared to those reported for
the enzymatic synthesis of enantiopure αꢀhydroxy compounds as
well as for several other kinetic resolutions, 54, 73, 81ꢀ83, 85 typically
5
4) and lipozyme (LM, Entry 5) were used in excessive amounts 55 ranging between 0.001 M and 0.3 M. The productivity and
for prolonged reaction times due to their very low activities in
10 standard conditions.
enantioselectivity is high due to the irreversibility of the
hydrolysis in excess of water and the straightforward conditions
of the biocatalytic system (absence of coꢀsubstrates and side
reactions). Figure 1 illustrates for instance the evolution of LꢀLA
60 production as a function of time for 0.55 M MLA at 45 °C in the
presence of CRL (triangles).
Improving the hydrolytic productivity
Because of the high enantiomeric excess of CRL, attempts were
made to maximize its volume productivity (expressed in g.Lꢀ1hꢀ1)
15 without compromising the excellent enantioselectivity. This is
essential, because productivity is a highly important measure for
industrial implementation.84 Attempts were made by inclining
Mechanistic discussion
A wide range of chiral esters and acids are nicely mapped in
literature with respect to the enantioselectivity in respectively
substrate, enzyme and buffer concentration, while maintaining 65 hydrolysis and esterification reactions and a predictive rule was
the same ratio of MLA to enzyme, together with an increase in
20 temperature. The effects on the volume productivity are
illustrated with catalytic data in Table 2.
established for CRL based on this data set. A true focus on αꢀ
hydroxy esters is however rather exceptional. Note that this rule
is less certain when dealing with crude CRL,86 which consists of
multiple isoforms differing in their enantioselectivity (see
70 supporting information).87ꢀ89 Moreover, studying a variety of the
substrate scope is a useful tool to gain insight into the catalytic
reactivity trends.90 Both the effects of variations at the acyl side,
viz. R‘ (Me and Et) directly connected with the chiral αꢀcarbon
and at the alkyl side R (Me, Et and nPr) were studied (see Table
75 2). To make a fair kinetic comparison, we limited our hydrolysis
study to waterꢀsoluble αꢀhydroxy esters. Note that the chiral
products of some substrates, viz. methyl αꢀhydroxy butyrate
(MHBA), have been suggested to be promising polymer
precursors themselves.91, 92 The biocatalytic hydrolysis results are
80 summarized in Table 3.
Table 2 Effects of temperature and concentration on the productivity and
enantioselectivity of Candida rugosa lipase.a
Entry Substr.
[M]
Time
[h]
Temp. Conv.(L) Prod. eep Pur.
[°C]
[%]b [gLꢀ1hꢀ1] [%]b [%]d
1
2
3
0.14c
0.55
0.27
46
23
6
25
25
45
73.8
75.0
72.8
0.2
1.9
3.3
92.8 96.4
91.8 95.9
91.5 95.8
4
5
6
0.55
1.10
0.55
8
16
6
45
45
55
65.7
63.8
49.3
4.4
4.1
4.8
90.9 95.5
88.7 94.4
74.7 87.4
Table 3 Hydrolysis of various αꢀhydroxy esters.a
a
Reactions carried out with 0.08 g enzyme preparation (unless stated
25 otherwise) with constant MLA to buffer ratio. b Determined by chiral GC
(see experimental and supporting information).
preparation was used. Molar enantiomeric purity on product side (see
c
Entry
R; R’
Time Conv.(L) Prod.
eep
Pur.
0.02 g enzyme
[%]b [g.Lꢀ1hꢀ1]c [%]b [%]d
d
[h]
supporting informaton).
1a
1b
1c
2a
3a
3b
3c
CH3;CH3
CH2CH3; CH3
CH2CH2CH3; CH3
CH3; CH2CH3
CH3; H
8
65.7
76.1
55.0
84.3
14.4e
25.7e
29.4e
4.4
18.1
33.0
12.3
0.19
0.39
0.48
90.9 95.5
88.7 95.4
26.4 63.2
>95 98.2
2.3
1.5
4
30 The results show a higher conversion rate with increasing
substrate concentration without loss in enantioselectivity (Entry
2 vs. Entry 1). The productivity is 0.22 and 1.95 g.Lꢀ1hꢀ1 for the
conversion of 0.14 M and 0.55 M MLA respectively with an eep
value above 90% in favor of Lꢀlactic acid formation. By
35 increasing the reaction temperature to 45 °C (Entry 3), 65.7%
conversion of LꢀMLA was reached within 8 hours corresponding
to a productivity of 4.4 g.Lꢀ1hꢀ1, indicating the enzyme is still
stable at this temperature. The enantioselectivity remains high
indicating that the uncatalyzed background hydrolysis is
40 negligible at 45 °C. A raise to 55 °C (Entry 6) lowered the
enantioselectivity, viz. from 91 to 75%, while the productivity
was only slightly higher. This lower eep is explained by a
significant contribution of nonꢀstereoselective background
hydrolysis. A reaction at 65 °C is not stereoselective and very
45 slow suggesting CRL deactivation at this reaction temperature
(data not shown). A careful comparison of Entry 3 with Entries
30
26
24
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
CH2CH3; H
CH2CH2CH3; H
a Hydrolysis at 45 °C, 0.55 M substrate, 0.057 M phosphate buffer (pH =
7.2) and 0.08 g CRL.b Determined by chiral GC (see experimental and
c
d
supporting information).
See supporting information.
Molar
85 enantiomeric purity on product side (see supporting information).e
Conversion here defined as total conversion of glycolate due to achirality.
No significant difference in eepꢀvalues of MLA (Entry 1a) and
ethyl lactate (EtLA) (Entry 1b) were obtained, while the
enantioselectivity seriously decreases for the propyl lactate ester
90 (nPrLA) (Entry 1c). A similar selectivity pattern was reported for
the hydrolytic kinetic resolution of βꢀborylated carboxylic esters
with CALB.93 The Lꢀenantiomer is preferred for the three lactate
4
and 5, reveals
a
maximum productivity and high
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MLA if 50% of the total amount of lactate ester is converted. As
45 no significant influence on the hydrolysis rate was observed,
alcohol product poisoning is excluded.
esters, which is according to the predictive Kazlauskas rule
assuming OH as medium sized substituent and CH3 at the αꢀ
carbon as large sized substituent (see scheme in Table 3).
The enantioselectivity significantly increases with
elongation of the acyl side of the ester, viz. R’ = Et as in the case
of MHBA (compare Entries 1a and 2a). This observation is in
agreement with the established empirical rule, which suggests
that the size of the substituents is crucial for the enantioselectivity
of CRL:78, 79, 86, 87, 94ꢀ98 a larger size difference between R’ and
10 OH, results in a higher enantioselectivity due to a larger
difference in free energy between the transition states of the two
enantiomers.
The lipase catalyzed hydrolysis is in principle a baseꢀ
catalyzed mechanism, viz. starting with a nucleophilic attack of
15 the serine oxygen to the carbonyl carbon of the ester. Therefore it
was highly unexpected for this enzymatic reaction to reveal a
higher hydrolysis rate with increasing alkyl length, viz. with
electron donating property (or basicity),99 as it contradicts the
reactivity of esters for the classic baseꢀcatalyzed hydrolysis.100ꢀ102
20 Indeed, the hydrolysis of MLA, ETLA and nPrLA by action of
OHꢀ has been studied and the authors observed a rate decrease
with increasing alkyl length of the released alcohol (see inset of
Figure 1).102, 103 The difference in reaction rate is also apparent by
comparing the kinetic profiles of MLA (triangle), ETLA
25 (squares) and nPrLA (diamonds) (Figure 1).
To further elaborate the reversed rate order, kinetic
parameters were measured according to MichaelisꢀMenten
kinetics, which is appropriate to apply for our reaction system.109
50 The KM and vmax values of crude CRL for the different lactate
esters (with R = Me, Et and nPr) are depicted in the supporting
information. The vmax is found to increase with increasing length
of R in the lactate ester, as illustrated in the inset of Figure 1. In
the applied conditions of Table 3 and Figure 1, viz. 0.55 M, the
55 reaction is thus carried out well above KM and thus near the
maximum hydrolysis rate of the enzyme (see supporting
information). This is beneficial, as the enzyme is „saturated“ with
substrate and thus works at maximum capacity. The above
observed rate order with the three lactate esters is therefore
60 determined by vmax, but now the question arises which step
5
determines vmax
.
A detailed study of the individual steps of the catalytic
cycle discloses a valid explanation of the intriguing reactivity
pattern, viz. MLA << ELA < nPrLA. Ester hydrolysis by CRL is
65 believed to proceed according to
a twoꢀstep mechanism:
transesterification (equation (1)) and hydrolysis (equation (2)).
First the active serine site of the catalytic triad is acylated,
involving the formation of the first tetrahedral intermediate
(ET1). During this acylation, the corresponding alcohol is
70 released, caused by a nucleophilic attack of the serine to the
carbonyl carbon. The second step is the deacylation of the
enzyme. This step involves the formation of the second
tetrahedral intermediate (ET2), caused by the introduction of a
water molecule. Finally, the corresponding acid is released,
75 recovering the catalytic triad in the original state.110
In the applied hydrolytic conditions, the first step, with the
removal of the alcohol, is in principle irreversible, thus
determining the enantioselectivity.86, 87, 111 The acylation step is
80 often suggested to be rateꢀdetermining, the hydrolysis rate thus
being in favor of the ester with the best leaving group, viz. the
shortest alcohol.87, 112 This is not according to our findings in case
of αꢀhydroxy esters: nPrLA has the highest reactivity.
Fig.1 Production of lactic acid in time and Eꢀvalue (see experimental
part), starting from MLA(▲), EtLA (■) and nPrLA (♦) using 0.08 g CRL,
0.55 M of lactate ester and 7 mL of a 0.057 M phosphate buffer. Inset:
30
reversed reactivity pattern for alkyl lactates (R = Me, Et, nPr) in
enzymatic (♦) and classic base (▲) catalyzed hydrolysis.102
A true understanding of the reversed reactivity pattern is
85 only possible by considering all elementary reaction steps of the
catalytic cycle. The extended mechanistic proposal is presented in
Figure 2. Step (1) of the mechanism is regarded as the association
of enzyme (E) and substrate (S), forming an enzymeꢀsubstrate
(ES) complex, as described for the mechanistically identical
90 serine proteases.113ꢀ115 The initial step is largely determined by
the KMꢀvalue, whereas vmax (and thus our rate order) is
determined in the subsequent steps. As the ES complex is
identical for the three lactate esters in step (5) to (8), the rate
determining step is determined either in step (2), (3) or (4)
95 (Figure 3). Step (2) presents a nucleophilic attack of the serine to
Significantly high volume productivities with ETLA and PrLA
instead of MLA, viz. 18.1 and 33.0 g.Lꢀ1hꢀ1 respectively versus
4.4 g.Lꢀ1hꢀ1 (MLA), were calculated from the graphs. In these
35 conditions, around 50mol% of lactic acid is reached starting from
ETLA in 6 hours, creating the desired equimolar ester/acid
composition, with a molar enantiomeric purity of 93%.
One hypothesis to explain the reverse reactivity might rely
on a more pronounced inhibitory effect of the formed methanol
40 on the enzyme when compared to ethanol and nꢀpropanol.104ꢀ108
An enzymatic hydrolysis of nPrLA was therefore carried out in
the same conditions of Table 3, but now in presence of 0.061 g of
methanol, which equals the quantity released in a reaction with
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the carbonyl carbon, with the formation of the tetrahedral
intermediate and stabilization of the negative oxygen by
hydrogen bonds in the oxyanion hole, comprised of a Gly and Ala
residue. According to the classic baseꢀcatalyzed hydrolysis, the
formation of such tetrahedral intermediate, is rateꢀdetermining.
as the leaving ability of the alcohol is generally accepted to be
35 rate determining.87, 112
Conclusions
5
In conclusion, we succeeded in performing a
productive enantioseparation of chemically
formed, racemic αꢀhydroxy compounds such
40 as lactate esters with commercial Candida
rugosa lipase in mild hydrolytic conditions.
The envisioned route comprises a unique
synergy between chemoꢀ and biocatalysis: the
Snꢀcatalyzed formation of racemic alkyl
45 lactates from common sugars like sucrose in a
green alcoholic solvent, followed by the
enantioselective hydrolysis with CRL. Clear
advantages compared to the current lactic acid
production process are: i) no gypsum waste, ii)
50 a high volume productivity, viz. up to 18.1 g.Lꢀ
1hꢀ1, iii) less demanding workup, and iv) coꢀ
generation of enantiopure Lꢀ and Dꢀlactic acid
with molar enantiomeric purity, sufficiently
high to foresee various PLA polymers ranging
55 from more hydrolysable forms to strong
stereocomplexes with melting points between
200°C and 265 °C (see supporting
information).117 In meanwhile, a fully novel
reactivity trend was encountered, pointing the
60 ester with the longer leaving alcohol as the
Fig.2 Propose extended mechanism of the hydrolysis of αꢀhydroxy esters with CRL, based on
reports of Hirohara et al., Nishiziwa et al., Buchwald et al. and Satoh et al.
The alcohol addition will thus be faster with electron
101
withdrawing R groups.100,
As our kinetic data show the
most reactive one. An unencountered rate determining
deprotonation step of the histidine residu by the alcoholate ion
proved to be the explanation for this unexpected pattern, while
intelligently modifying the histidine basicity might result in an
65 increased intrinsic activity. An economic assessment based on the
obtained productivity, industrial product market prices and
reported enzyme stability, in similar reaction conditions, suggests
a profitable route towards PLA for specialty and medical
applications (see supporting information).118ꢀ123 As enzyme
70 stability and productivity were key in the process economics
evaluation, the study of immobilization strategies and protein
engineering will be crucial in subsequent research. A realistic
prospect based on recent achievements for analogue cases (see
supporting information) renders the proposed kinetic resolution
75 strategy challenging, but likely also for commodity applications.
opposite reactivity, step (2) is certainly not the rateꢀdetermining
step in the hydrolysis of lactates by CRL. Some literature
10 suggests that the formation rate of hydrogen bonds in the
oxyanion hole is defined sterically,116 but also according to this
hypothesis our results show the opposite trend. Next, the
tetrahedral intermediate collapses, with the release of ROH. This
collapse is often regarded as a one elementary step, in which the
15 best leaving alcohol determines the reactivity.87, 112 However, our
kinetic results agree with a 2ꢀstep interpretation of the collapse,
here presented by step (3) and (4). In step (3), the alcoholate ion
leaves the acylated serine, we propose that ROꢀ binds the
histidine proton in accordance with other reports.110, 116 Step (4)
20 requires a deprotonation of the histidine with release of ROH,
permitting the introduction of a water molecule to initiate the
hydrolysis sequence in step (5ꢀ8), ultimately resulting in the acid
production. Our reactivity pattern is in accord with a rate
determining histidine deprotonation (step (4)): the more electron
25 donating the alcoholate, the better it accepts the histidine proton.
Following this theory, a clear explanation for the reactivity
differences of nPrLA, ETLA and MLA is provided.
Notes and references
a Center for Surface Chemistry and Catalysis, K. U. Leuven, Kasteelpark
Arenberg 23, 3001 Heverlee, Belgium. Fax: (+)32 16321998; Tel: (+)32
16 3 21593; E-mail: bert.sels@biw.kuleuven.be
80 † Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
‡ P.V.W. acknowledges the Research Council of the K.U. Leuven (IDO
ꢀ 3E090504), M.D. thanks “FWO Vlaanderen” (Grant 1.1.955.10N).
85 Methusalem and IAP (Belspo) are acknowledged for financial support.
Purac is also gratefully thanked for providing lactate and lactide samples.
To verify our hypothesis for other αꢀhydroxy esters, we
additionally investigated the hydrolysis of glycolates, the
30 shortest αꢀhydroxy esters, with CRL. The reaction kinetics follow
the same rate order (compare entries 3a-c in Table 3) as for the
lactates. The observation of
a rate determining histidine
deprotonation in a lipase catalyzed hydrolysis of esters is unique
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